AGRIN RECEPTORS IN NEURAL DEVELOPMENT

AGRIN 受体在神经发育中的作用

基本信息

批准号：
2519772
负责人：
JUSTIN R. FALLON
金额：
$ 16.44万
依托单位：
BROWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-09-30 至 1999-08-31
项目状态：
已结题

项目摘要

Proper development and functioning of the brain relies on the precise and rapid flow of signals between neurons. Synapses play the foremost role in this information exchange. While the physiology and structure of mature synapses are understood in considerable depth, much less is known about the molecular cues mediating synaptic differentiation, maintenance, and turnover. A cardinal event in synapse formation is the marshalling of neurotransmitter receptors to the postsynaptic membrane. Mounting evidence suggests that agrin, a protein that directs postsynaptic differentiation at developing neuromuscular junctions, may also play a role in synaptogenesis in the brain. A full understanding of agrin's function in the brain requires the characterization of its cell surface receptor. In preliminary experiments we have found that agrin binds to cultured neurons and to synaptosomes. In some cells, agrin elicits the redistribution of these binding sites, suggesting that they constitute functional agrin receptors. Further, we have identified two agrin binding polypeptides in brain membranes. In the proposed studies we will characterize neuronal agrin receptor(s) at the cellular, biochemical, and functional levels, with the overall goal of elucidating the molecular events underlying synapse formation among neurons. Using well characterized primary hippocampal cultures, we will determine the time course of expression, the cellular localization, and the functional properties of agrin receptors on differentiating neurons. We will compare the distribution of agrin receptors to that of neurotransmitter receptors and other synaptic specializations. The subcellular localization of agrin receptors will be analyzed at the ultrastructural level. We will also determine whether agrin induces changes in the distribution of its own receptor or of related markers of synaptic differentiation. In parallel with this cell biological approach, we will characterize neural agrin receptors biochemically. Ligand overlay techniques and affinity chromatography methods will be used to identify and to determine the subunit composition of agrin receptors on neurons, and partial amino acid sequence of the purified receptors will be obtained. In addition, we will determine how alternative splicing of agrin may regulate binding to agrin receptors, and investigate the ensuing functional consequences. The results of these experiments will provide fundamental insights into synapse formation, modification, loss, and recovery. Knowledge of these basic mechanisms is crucial to our understanding of normal synaptic function, and forms a bedrock for the development of diagnostic and therapeutic approaches aimed at ameliorating or curing mental illnesses.

大脑的正常发育和功能依赖于精确和神经元之间的信号快速流动。突触在其中起着最重要的作用这种信息交换。当生理和结构成熟时人们对突触的了解相当深入，但对突触的了解却少之又少介导突触分化、维持的分子线索周转。突触形成的一个主要事件是编组突触后膜的神经递质受体。越来越多的证据表明集聚蛋白（agrin）是一种指导突触后分化的蛋白质在神经肌肉接头的发育中，也可能发挥作用大脑中的突触发生。全面了解 agrin 的功能大脑需要对其细胞表面受体进行表征。在初步实验我们发现集聚蛋白与培养的神经元结合和突触体。在某些细胞中，集聚蛋白会引发这些结合位点，表明它们构成了功能性集聚蛋白受体。此外，我们还鉴定了两种集聚蛋白结合多肽脑膜。在拟议的研究中，我们将表征神经元集聚蛋白受体细胞、生化和功能水平，总体目标是阐明突触形成的分子事件神经元。使用特征明确的原代海马培养物，我们将确定表达的时间过程、细胞定位，以及集聚蛋白受体对分化神经元的功能特性。我们将比较集聚蛋白受体的分布神经递质受体和其他突触专门化。这集聚蛋白受体的亚细胞定位将在超微结构水平。我们还将确定集聚蛋白是否会诱导其自身受体或相关标记物的分布发生变化突触分化。与这种细胞生物学方法并行，我们将从生化角度表征神经集聚蛋白受体。配体叠加技术和亲和层析方法将用于鉴定并确定神经元上集聚蛋白受体的亚基组成，纯化的受体的部分氨基酸序列为获得。此外，我们将确定集聚蛋白的选择性剪接如何可能调节与集聚蛋白受体的结合，并研究随后的功能性后果。这些实验的结果将提供基本的见解突触的形成、修饰、丢失和恢复。这些知识基本机制对于我们理解正常突触至关重要功能，并为诊断和诊断的发展奠定了基础旨在改善或治愈精神疾病的治疗方法。